A current major challenge in pharmaceutical industry is to develop treatments for brain diseases and mental disorders such as depression, anxiety, schizophrenia, epilepsy, addiction and many more. Over the last decade, the drug discovery process has evolved dramatically following the continued advancement of technologies. In addition to increasing the efficiency of high-throughput screening (HTS) by assay miniaturization and multiplexing, various initiatives have been taken to improve the science of predicting toxicity and improving extrapolation to humans. With recent advances in genomics and proteomics, a number of novel technologies have made the utilization of live cells with overexpressed drug targets in assays an important aspect in the drug discovery process. Nevertheless, an appropriate cellular platform for HTS of neural targets has not been available. This in part can be attributed to difficulties in transfecting and maintaining neurons in culture. Recently, a breakthrough was made in human embryonic stem cell research that allows in vitro differentiation of neurons from a renewable source of progenitor cells. However, limitations of the current differentiation protocol and poor neuroprogenitor cell transfection techniques have restricted the use of these cells in assays. Originus, Inc. can contribute to the development of sophisticated neuronal cell-based assay platforms that address these issues by using a proprietary solid phase cell transfection technology termed Surface Transfection and Expression Protocol (STEP) exclusively licensed from the University of Michigan. We have successfully developed multiple G- protein-coupled receptor (GPCR) assays in neuronal and non-neuronal cell lines using STEP. The proposed new STEP transfection and differentiation platform will drastically accelerate human stem cell neural differentiation process while simultaneously introducing the target of interest and necessary components for a cell-based assay. Our goal is to develop not only more reliable and robust assays that increase the screening efficiency in early preclinical phase, but also more physiologically relevant assays that may boost clinical success rates. During Phase I of this application we will initially examine the biological responses of the neural progenitor cells in assays, and thoroughly characterize the progenitor cell neuronal differentiation induced by transient over-expression of selected basic helix-loop-helix (bHLH) transcription factors. Further, the potential implication of such a platform for cell-based assays will be demonstrated by examining the response of metabotropic glutamate receptors in differentiated human neurons. During the Phase II, we plan to apply this technology to other human pluripotent cell lines, expand the spectrum of bHLH factors tested to fine tune the differentiation process, and to further develop assays for different drug targets. This novel platform will make feasible HTS of drug targets expressed in human neurons. In addition, it may also be used for toxicology, neural differentiation and degeneration studies. PUBLIC HEALTH RELEVANCE: The proposed studies aim to greatly improve the effectiveness of the drug discovery process for brain diseases and mental disorders by taking advantage of research advances in human stem cells and genetic control of neuronal development. Specifically, the goal of this application is to develop physiologically relevant drug screening platforms for human neurons derived from embryonic stem cells.